Drug conjugates

ABSTRACT

A compound having the formula Y-A-Z, wherein: 
     A is a 5, 6, or 7 member ring that is monocyclic or is fused to 1 to 3 additional 4 to 8 member rings; wherein ring A and, independently, the fused additional rings are carbocyclic or heterocyclic, and saturated or unsaturated, wherein unsaturated rings are aromatic or non-aromatic; wherein Y and Z are substituents at adjacent positions on ring A; 
     Y represents: 
     
       
         
         
             
             
         
       
     
     Z represents: 
     
       
         
         
             
             
         
       
     
     X and E represent O, S, or NR a  or NR b ; each of a, b, c, d, e and f independently represents 0 or 1; a+c equals 0, 1, or 2; b+d equals 0, 1, or 2; a+b+c+d+e+f equals 1, 2, or 3; provided that when f is 1, then d is 1, and when d is 0, then f is 0; and when both e and b are 0, then neither R 1  nor R 2  is chloro or bromo; v represents 0 or 1, provided that when v is 0, then J is hydrogen, a metal ion, or a quaternary ammonium ion, and X is O and G is H; 
     either G is hydrogen, a metal ion, a quaternary ammonium ion, lower alkyl, or comprised of a pharmaceutically active chemical compound or the precursor thereof; or X-G represents a carbonyl-activating group; 
     J is lower alkyl, aryl, heteroaryl, omega-hydroxycarbonyl-(lower alkyl), omega-(lower alkoxy)carbonyl-(lower alkyl), omega-(X-G)-carbonyl-(lower alkyl) group, or comprised of a specific binding agent; and R a , R b , R 1 , R 2 , R 3 , R 4  are as defined in the specification.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 60/544,033, filed on Feb. 12, 2004, and U.S. application Ser. No.11/053,655, filed on Feb. 8, 2005. Both of these applications are herebyincorporated by reference in their entireties.

The present invention was made with government support under Grant No.R01 GM427980 as well as R01CA103314 awarded by the National Institutesof Health. The United States government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

The advantage of drugs that are capable of selectively targeting aspecific cell type is well recognized. Thus, designing such selectivedrugs is a field of intense study. Such drugs are often, but by no meansalways, anticancer drugs.

Of importance in this regard, are drugs (or pro-drugs) that are bound toa marker. The purpose of the marker is to direct the drug to amarker-specific target cell. For example, binding drugs to antibodiesfor the purpose of targeting antibody-specific cells, such as tumorcells, is well known.

Binding a marker directly to a drug is, however, not always feasible. Inaddition, binding a marker directly to a drug often has the effect ofdramatically reducing the efficacy of the drug, or disabling the actionof the drug completely.

One approach to reducing the loss of efficacy of a drug bound to amarker is to bind the marker to the drug in a way that causes the drugto be released at the target in its most active form (usually the parentdrug itself. In this regard, there has been intense interest in findingheterobifunctional compounds, or linkers, that not only bind a markerand a drug, but that also cause the drug to be released in its mostactive form at the appropriate time.

For example, U.S. Pat. No. 4,880,935 to Thorpe and U.S. Pat. No.5,936,092 to Shen, et al. disclose heterobifunctional compounds thatcontain a disulfide group at one end and a carboxylate group at theother end. A cytotoxin is bound to the disulfide group at one end. Anantibody is bound to the carboxylate group at the other end. Once in thetargeted cell, the disulfide bond is cleaved by endogenousdisulfide-reducing peptides, such as glutathione (GT). Cleavage of thedisulfide bond releases the parent cytotoxin inside the targeted cell.

Similarly, U.S. Pat. No. 5,137,877 to Kaneko, et al., discloseheterobifunctional compounds that contain a hydrazinyl group at one endand a disulfide group at the other end. An anthracycline-based cytotoxinis bound to the hydrazinyl group. An antibody is bound to the disulfidegroup. The more acidic environment of the target cells causes hydrolysisof the hydrazinyl group. Hydrolysis of the hydrazinyl group releases theparent cytotoxin.

However, a demonstrated release of the drug at a target does notnecessarily make the heterobifunctional compound medically effective ordesirable. Medical efficacy depends to a significant extent on the rateof release of the drug by the heterobifunctional compound.

Accordingly, there have been efforts to increase the rates of release ofdrugs at a specified target by designing new heterobifunctionalcompounds. Most notable are efforts to design heterobifunctionalcompounds that, when hydrolyzed, undergo a favorable cyclizationreaction to release the drug.

For example, Y. Ueda et al., Bioorganic & Medicinal Chemistry Letters,1993, Vol. 3, No. 8, 1761-1766, disclose linkers that undergophosphatase-inititiated lactonization of phosphonoxyphenylpropionatederivatives of Taxol. The lactonization results in the in vivo releaseof the parent Taxol. As shown in Ueda et al., the purpose of Ueda'sphosphonoxyphenylpropionate group is to increase the water solubility ofTaxol.

R. B. Greenwald et al., J. Med. Chem., 2000, 43, 475-487, discloseester-containing compounds linked to a drug to form a pro-drug. Thepro-drug is hydrolyzed by an esterase, which results in a lactonizationreaction. The lactonization reaction releases the drug.

K. Achilles, Arch. Pharm. Pharm. Med. Chem., 2001, 334, 209-215,discloses esterase-initiated lactonization of heterobifunctionalcompounds containing a peptide binding agent and a pro-drug. Thelactonization causes release of the drug. The peptides bind topolymorphonuclear elastase, a serine protease associated with numerousmedical conditions, including cancer.

Similarly, B. Wang et al. J. Org. Chem., 1997, 62, 1363-1367, discloseesterase-initiated lactonization of prodrugs containing cyclic peptidesas binding agents.

However, the efficacy of conjugates of the heterobifunctional compoundsdescribed above is, in the vast majority of cases, far from clinicallyuseful. For example, it would be particularly beneficial to increase therates of drug release of such conjugates in order to be clinicallyeffective.

Such targeted and effective rates of release of drugs have not yet beenrealized. In the case of targeting tumor cells, such high rates ofrelease of anti-cancer drugs are particularly critical in light of theknown high proliferation of cancer cells in tumors.

In addition, the drug conjugate containing a linker should release themost active form of the drug. See I. Ojima, X. Geng, X. Wu, C. Qu, C. P.Borella, H. Xie, S. D. Wilhelm, B. A. Leece, L. M. Bartle, V. S.Goldmacher, and R. V. J. Chari, J. Med. Chem. 45, 5620-5623 (2002).

Taxol, a diterpene natural product, has gained prominence as one of themost efficacious anticancer drugs. See E. K. Rowinsky, Annual Review ofMedicine 1997, 48, 353; M. Suffness, Taxol Science and Applications; CRCPress: New York, 1995. Even more promising congeners of Taxol, termedtaxoids (Taxol-like compounds), with orders of magnitude higher potencythan Taxol have been developed. See G. I. Georg, T. Chen, I. Ojima, andD. M. Vyas (Eds.),“Taxane Anticancer Agents: Basic Science and CurrentStatus”, ACS Symp. Series 583; American Chemical Society, Washington, D.C., 1995); I. Ojima, et al, Bioorg. Med. Chem. Lett., 1999, 9,3423-3428; I. Ojima, et al, J. Med. Chem., 1996, 39, 3889-3896; and I.Ojima, G. D. Vite, K. -H. Altmann (Eds.), “Anticancer Agents: Frontiersin Cancer Chemotherapy”, ACS Symp. Series 796, American ChemicalSociety, Washington, D. C., 2001.

However, Taxol, taxoids, and other cytotoxic anticancer drugs containdrawbacks, including high toxicity to normal cells, poor watersolubility, and emergence of drug-resistance. Therefore, there has beenparticular interest in selectively and efficiently targeting tumor cellswith cytotoxic anticancer drugs. See, for example, K. Achilles, Arch.Pharm. Pharm. Med. Chem., 2001, 334, 209-215, and U.S. Pat. No.5,137,877 to Kaneko, et al. R. V. J. Chari, Advanced Drug DeliveryReviews, 1998, 31, 89-104; M. L. Disis and M. A. Cheever, Advances inCancer Research, 1997, 71, 343-371; H. Bier, T. Hoffmann, I. Haas, A.Van Lierop, Cancer Immunology Immunotherapy, 1998. 46, 167-173; G. A.Pietersz, B. Toohey, l. F. C. McKenzie, Journal of Drug Targeting, 1998,5, 109-120; P. R. Hamann, L. M. Hinman, I. Hollander, C. F. Beyer, D.Lindh, R. Holcomb, W. Hallett, H. -R. Tsou, J. Upeslacis, D. Shochat, A.Mountain, D. A. Flowers, I. Bernstein, Bioconjugate Chemistry, 2002, 13,47-58; Firestone, R. A.; Dubowchik, G. M. In Eur. Pat. Appl. EP 0624377(Bristol-Myers Squibb Co. USA)1994; A. Safavy, K. P. Raisch, M. B.Khazaeli, D. J. Buchsbaum, J. A. Bonner, Journal of Medicinal Chemistry,1999,42, 4919-4924; C. Li, D. Yu, T. Inoue, D. J. Yang, L. Milas, N. R.Hunter, E. E. Kim, S. Wallace, Anti-Cancer Drugs, 1996, 7, 642-648;Pendri, A.; Conover, C. D.; Greenwald, R. B. Anti-Cancer Drug Design,1998, 13, 387; C. Li, D. -F. Yu, R. A. Newman, F. Cabral, L. C.Stephens, N. Hunter, L. Milas, S. Wallace, Cancer Research, 1998, 58,2404-2409; W. C. Rose, J. L. Clark, F. Y. F. Lee, A. M. Casazza, CancerChemotherapy and Pharmacology, 1997, 39, 486-492; J. J. Correa, M. Page,Tumor Targeting in Cancer Therapy, 2002, 165-178; V. Guillemard, H. U.Saragovi, Cancer Research, 2001, 61, 694-699; I. Ojima, X. Geng, X. Wu,C. Qu, C. P. Borella, H. Xie, S. D. Wilhelm, B. A. Leece, L. M. Bartle,V. S. Goldmacher, and R. V. J. Chari, J. Med. Chem. 45, 5620-5623(2002).

Thus, there is a need for improving, inter alia, the specificity of drugdelivery and the rates of release of various of drugs, includinganticancer drugs. To achieve the above, there is a need for improvedheterobifunctional linkers that bind a drug to a cell-specific marker,and that release the most active form of the drug in the target cell.There is a particular need for heterobifunctional linkers thatselectively target tumor cells with highly potent cytotoxic drugs at adrug release rate that is effective for the destruction and/orinhibition of tumor cells.

SUMMARY OF THE INVENTION

These, and other objectives as will be apparent to those of ordinaryskill in the art, have been achieved by providing a compound having theformula Y-A-Z. wherein:

-   -   A is a 5, 6, or 7 member ring that is monocyclic or is fused to        1 to 3 additional 4 to 8 member rings; wherein ring A and,        independently, the fused additional rings are carbocyclic or        heterocyclic, and saturated or unsaturated, wherein unsaturated        rings are aromatic or non-aromatic;    -   ring A and the additional rings are unsubstituted or substituted        with 1 to 4 substituents selected from lower alkyl, aryl,        heteroaryl, hydroxy-(lower alkyl), amino-(lower alkyl), N-(lower        alkyl)amino-(lower alkyl), N,N-di(lower alkyl)amino-(lower        alkyl), N-arylamino-(lower alkyl), N,N-diarylamino-(lower        alkyl), N-(heteroaryl)amino-(lower alkyl),        N,N-di(heteroaryl)amino-(lower alkyl), hydroxylamino, O-(lower        alkoxy)amino, O-aryloyamino, O-heteroaryloxyamino, fluoro,        chloro, bromo, nitro, hydroxyl, lower alkoxy,, aryloxy, carboxyl        (hydroxycarbonyl), lower alkanoyl, lower alkanoyloxy, amino,        N-lower alkyl)amino, N,N-di(lower alkyl)amino, formamido        (formylamino), N-acylamino, N,N-diacylamino (imido), hydrazido,        N-(lower alkyl)hydrazido, N,N-di(lower alkyl)hydrazido,        N-arylhydrazido, N,N-diarylhydrazido, N-(heteroaryl)hydrozido,        N,N-di(heteroaryl)hydrazido, carboxamido (carbamoyl), N-(lower        alkyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl,        N,N-diarylcarbamoyl, N-heteroarylcarbamoyl,        N,N-di(heteroaryl)carbamoyl, hydroxysulfonyl (sulfonic acid),        (lower alkoxy)sulfonyl, aryoxysulfonyl, heroaryloxysulfonyl,        hydroxysulfonyl-(lower alkyl), (lower alkoxy)sulfonyl-(lower        alkyl), aryoxysulfonyl-(lower alkyl), heroaryloxysulfonyl-(lower        alklyl), (lower alkane)sulfonyl, arenesulfonyl, or        heteroarenesulfonyl;    -   Y and Z are substituents at adjacent positions on ring A;    -   Y represents:

-   -   Z represents:

-   -   X represents O, S, or NR^(a);    -   E represents O, S, or NR^(b);    -   R^(a) and R^(b) independently represent H, lower alkyl,        cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl,        aryl, aryl lower alkyl, heteroaryl, lower alkanoyl, aromatic        acyl (arenecarbonyl), heteroaromatic acyl (heteroarenecarbonyl),        (lower alkoxy)carbonyl, aryloxycarbonyl, heteroaryloxycarbonyl,        N-arylcarbamoyl, N,N-diarylcarbamoyl, N-heteroarylcarbamoyl,        N,N-di(heteroaryl)carbamoyl, (lower alkane)sulfonyl,        arenesulfonyl, or heteroarenesulfonyl, wherein the lower alkyl,        lower alkanoyl and lower alkoxy groups are unsubstituted or        substituted with 1 to 4 groups independently selected from aryl,        heteroaxyl or fluoro;    -   each of a, b, c, d, e and f independently represents 0 or 1;    -   a+c equals 0, 1, or 2;    -   b+d equals0, 1,or2;    -   a+b+c+d+e+f equals 1,2, or3;    -   provided that when f is 1, then d is 1, and when d is 0, then f        is 0; and when both e and b are 0, then neither R¹ nor R² is        chloro or bromo.    -   v represents 0 or 1, provided that when v is 0, then J is        hydrogen, a metal ion, or a quaternary ammonium ion; and X is O        and G is H;    -   each of R¹ and R² independently represents H, lower alkyl, aryl,        heteroaryl, fluoro, chloro, bromo, lower alkoxy (i.e.,        alkyloxy), aryloxy, heteroaryloxy, N,N-di(lower alkyl)amino,        N,N-diarylamino, N,N-di(heteroaryl)amino, (lower alkyl)thio,        arylthio, heteroarylthio, (lower alkane)sulfinyl, arenesulfinyl,        heteroarenesulfinyl, (lower alkyl)sulfonyl, arenesulfonyl,        heteroarenesulfonyl, (lower alkoxy)-(lower alkyl),        aryloxy-(lower alkyl), heteroaryloxy-(lower alkyl), N,N-di(lower        alkyl)amino-(lower alkyl), N,N-diarylamino-(lower alkyl),        N,N-di(heteroaryl)amino-(lower alkyl), (lower alkyl)thio-(lower        alkyl), arylthio-(lower alkyl), heteroarylthio-(lower alkyl),        (lower alkane)sulfinyl-(lower alkyl), arenesulfinyl-(lower        alkyl), heteroarenesulfinyl-(lower alkyl), (lower        alkyl)sulfonyl-(lower alkyl), arenesulfonyl-(lower alkyl),        heteroarenesulfonyl-(lower alkyl), (lower alkoxy)carbonyl,        aryloxycarbonyl, heteroaryloxycarbonyl, carboxamido (carbamoyl),        N-(lower alkyl)carbamoyl, N,N-(lower alkyl)carbamoyl,        N-arylcarbamoyl, N,N-diarylcarbamoyl, N-heteroarylcarbamoyl or        N,N-di(heteroaryl)carbamoyl;    -   each of R³ and R⁴ independently represents H, lower alkyl, aryl,        heteroaryl, fluoro, lower alkoxy, aryloxy, heteroaryloxy,        N,N-di(lower alkyl)amino, N,N-diarylamino,        N,N-di(heteroaryl)amino, (lower alkyl)thio, arylthio,        heteroarylthio, (lower alkane)sulfinyl, arenesulfinyl,        heteroarenesulfinyl, (lower alkyl)sulfonyl, arenesulfonyl,        heteroarenesulfonyl, (lower alkoxy)-(lower alkyl),        aryloxy-(lower alkyl), heteroaryloxy-(lower alkyl), N,N-di(lower        alkyl)amino-(lower alkyl), N,N-diarylamino-(lower alkyl),        N,N-di(heteroaryl)amino-(lower alkyl), (lower alkyl)thio-(lower        alkyl), arylthio-(lower alkyl), heteroaxylthio-(lower alkyl),        (lower alkane)sulfinyl-(lower alkyl), arenesulfinyl-(lower        alkyl), heteroarenesulfinyl-(lower alkyl), (lower        alkyl)sulfonyl-(lower alkyl), arenesulfonyl-(lower alkyl),        heteroarenesulfonyl-(lower alkyl), (lower alkoxy)carbonyl,        aryloxycarbonyl, heteroaryloxycarbonyl, carboxamido (carbamoyl),        N-(lower alkyl)carbamoyl or N,N-(lower alkyl)carbamoyl,        N-arylcarbamoyl, N,N-diarylcarbamoyl, N-heteroarylcarbamoyl or        N,N-di(heteroaryl)carbamoyl;    -   R¹ and R² or R³ and R⁴ are optionally connected to form a 3 to 8        member ring, wherein the 3 to 8 member ring is cycloalkyl,        heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, or        heteroaryl;    -   lower means having 1-6 carbon atoms;    -   either G is hydrogen, a metal ion, a quaternary ammonium ion,        lower alkyl, or comprised of a pharmaceutically active chemical        compound or the precursor thereof; or X-G represents a        carbonyl-activating group;    -   J is lower alkyl, aryl, heteroaryl, omega-hydroxycarbonyl-(lower        alkyl), omega-(lower alkoxy)carbonyl-(lower alkyl),        omega-(X-G)-carbonyl-(lower alkyl) group, or comprised of a        specific binding agent.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a drug conjugate having the formula Y-A-Z. Inthe formula, A is a 5, 6, or 7 member ring. In one embodiment, ring A ismonocyclic and carbocyclic. The monocyclic, carbocyclic ring may besaturated. Some examples of suitable saturated rings includecyclopentane, cyclohexane, and cycloheptane rings.

Alternatively, the monocyclic carbocyclic ring may be unsaturated. Theunsaturated ring contains at least one double bond. For example, a 5member ring can have 1 or 2 double bonds, and a 7 member ring can have 1to 3 double bonds. The unsaturated rings may be aromatic, i.e., “aryl”or “arene,” or non-aromatic. Some examples of suitable unsaturatedmonocyclic carbocyclic rings include cyclopentene, cyclohexene,cycloheptene, cyclopentadiene, 1,3-cyclohexadiene, 1,4-cyclohexadiene,1,3-cycloheptadiene, and cycloheptatriene rings. Preferably, theunsaturated carbocyclic ring is a benzene ring, i.e., phenylene.

In another embodiment, ring A is a heterocyclic ring. The heterocyclicring contains at least one, and up to four, of any of the heteroatomsnitrogen (N), oxygen (O), or sulfur (S), or any combination thereof. Thenitrogen and sulfur heteroatoms may also contain additionalsubstituents. For example, a nitrogen heteroatom may be an amine oxide,oxime, O-(lower alkyl)-oxime, hydroxylamine, O-(loweralkyl)-hydroxylamine, hydrazine, N-(lower alkyl)-hydrazine, N,N-di(loweralkyl)-hydrazine, N-arylhydrazine, N,N-diarylhydrazine, or hydrazone;and a sulfur heteroatom may be a sulfoxide or sulfone. In addition,where the nitrogen is not double bonded within the ring, the nitrogenmay be substituted by H, lower alkyl, cycloalkyl, heterocycloalkyl,cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, lower alkanoyl,arenecarbonyl, heteroarenecarbonyl, (lower alkoxy)carbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, carboxamido (carbamoyl),N-(lower alkyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl,N,N-diarylcarbamoyl, N-heteroarylcarbamoyl, N,N-di(heteroaryl)carbamoyl,(lower alkane)sulfinyl, arenesulfinyl, heteroarenesulfinyl, (loweralkane)sulfonyl, arenesulfonyl, or heteroarenesulfonyl.

In one embodiment, the heterocyclic ring may be saturated. Some examplesof suitable saturated heterocyclic rings containing a single nitrogenheteroatom include pyrrolidine, piperidine, homopiperidine (azepane),N-methylpyrrolidine, N-(tert-butoxycarbonyl)pyrrolidine,N-acetylpyrrolidine, pyrrolidine N-oxide, pyrrolidine N-hydroxide,pyrrolidine N-methoxide, N-methylpiperidine, N-acetyl piperidine,piperidine N-oxide, piperidine N-hydroxide, piperidine N-methoxide, andN-benzylpiperidine rings.

Some examples of suitable saturated heterocyclic rings containing morethan one nitrogen heteroatom include imidazolidine,N,N′-dimethylimidazolidine, pyrazolidine, piperazine,1-acetylpiperazine, 1-(o-tolyl)piperazine, piperazine-N,N′-dioxide,tert-butyl4-benzyl-1-piperazinecarboxylate, 1-benzylpiperazine, benzyl1-piperazinecarboxylate, hexahydropyrimidine, homopiperazine(1,4-diazepane), tert-butyl 1-homopiperazinecarboxylate, benzyl1-homopiperazinecarboxylate, hexahydro-1,3,5-triazine(1,3,5-triazinane), 1,3,5-trimethylhexahydro-1,3,5-triazine,1,3,5-triacryloylhexahydro- ,3,5-triazine, and I,3,5-tribenzylhexahydro-1,3,5-triazine rings.

Some examples of suitable saturated heterocyclic rings containing asingle oxygen heteroatom include tetrahydrofuran, tetrahydropyran, andoxacycloheptane (oxepane) rings. Some examples of suitable saturated 5,6, or 7 member heterocyclic rings containing more than one oxygenheteroatom include 1,3-dioxolane, 1,3-dioxane, 1,4-dioxane, and1,3-dioxepane rings.

Some examples of suitable saturated heterocyclic rings containing asingle sulfur heteroatom include tetrahydrothiophene,tetrahydrothiopyran, thiacycloheptane (thiepane), tetrahydrothiophene1-oxide, tetrahydrothiophene-1,1-dioxide (tetramethylene sulfone, orsulfolane), and tetrahydrothiopyran-1,1-dioxide rings. Some examples ofsuitable saturated 5, 6, or 7 member heterocyclic rings containing morethan one sulfur heteroatom include 1,3-dithiolane, 1,3-dithiane,1,4-dithiane, 1,3-dithiepane, 1,1,3,3-tetramethyl-1,3-dithiolane, and1,1,4,4-tetramethyl-1,4-dithiane rings.

Some examples of suitable saturated heterocyclic rings containing acombination of 2 to 4 heteroatoms include 1,3-oxazolidine,N-butyl-oxazolidine, 1,3-thiazolidine, 1,3-oxathiolane, morpholine,thiomorpholine (1,4-thiazinane), 4-acetylmorpholine, 1,4-oxathiane,1,3-oxazinane, 1,3-thiazinane, 4-aza-1-oxa-cycloheptane (1,4-oxazepane),1,3,4-oxadiazolidine, 1,3,5-oxadiazinane, 1,3,2-dioxathiolane2,2-dioxide, and 1-oxahexahydro-3,4,5-triazine (1-oxa-3,4,5-triazinane)rings.

Alternatively, the heterocyclic ring may be unsaturated. The unsaturatedheterocyclic rings may be aromatic, i.e., “heteroaryl” or “heteroarene,”or non-aromatic. Some examples of suitable unsaturated heterocyclicrings containing a single nitrogen heteroatom include 1H-pyrrole,2,5-dihydro-1H-pyrrole, pyridine, pyridine N-oxide,1-aza-2,4,6-cycloheptatriene (azepine), tert-butyl2,5-dihydro-1H-pyrrole-1-carboxylate, 1-(p-toluenesulfonyl)pyrrole, and4-(benzyloxy)pyridine N-oxide rings. Some examples of suitableunsaturated heterocyclic rings containing more than one nitrogenheteroatom include imidazole, 1H-pyrazole, pyrazine, pyrimidine,1,3,5-triazine, 1H-1,2,3-triazole, 1H-1,2,4-triazole,1-(p-toluenesulfonyl)imidazole, 1-acetylimidazole,1-(tert-butoxycarbonyl)imidazole, and 1,1′-sulfonyldiimidazole rings.

Some examples of suitable unsaturated heterocyclic rings containing asingle oxygen heteroatom include furan, 2,5-dihydrofuran,2,3-dihydrofuran, 2H-pyran, 4H-pyran, 3,4-dihydro-2H-pyran, and1-oxa-2,4,6-cycloheptatriene (oxepine) rings. Some examples of suitableunsaturated heterocyclic rings containing more than one oxygenheteroatom include 1,3-dioxacyclopent-4-ene (1,3-dioxole),1,3-dioxacyclohex-4-ene (4H-1,3-dioxine), 1,4-dioxacyclohex-2-ene(2,3-dihydro-1,4-dioxine), and 1,4-dioxacyclohexa-2,5-diene(1,4-dioxine) rings.

Some examples of suitable unsaturated heterocyclic rings containing asingle sulfur heteroatom include thiophene, thiophene-1,1-dioxide,2,5-dihydrothiophene, 2,5-dihydrothiophene-1-oxide (butadienesulfoxide), 2,5-dihydrothiophene-1,1-dioxide (butadiene sulfone),2,3-dihydrothiophene, 2H-thiopyran, 4H-thiopyran,3,4-dihydro-2H-thiopyran, 4H-thiopyran-1,1-dioxide, and1-thia-2,4,6-cycloheptatriene (thiepine) rings. Some examples ofsuitable unsaturated 5, 6, or 7 member heterocyclic rings containingmore than one sulfur heteroatom include 1,3-dithiacyclopent-4-ene(1,3-dithiole), 1,3-dithiacyclohex-4-ene (4H-1,3-dithiine),1,4-dithiacyclohex-2-ene (2,3-dihydro-1,4-dithiine),1,3-dithiacyclohept-5-ene (4,7-dihydro-1,3-dithiepine), and1,4-dithiacyclohexa-2,5-diene (1,4-dithiine) rings.

Some examples of suitable unsaturated heterocyclic rings containing acombination of 2 to 4 heteroatoms, include oxazole, thiazole, oxathiole,2H-1,4- oxazine, 4H-1,4-oxazine, 2H-1,3-oxazine,1,4-oxazine-4-carboxylic acid tert-butyl ester, 1,4-oxazine-4-carboxylicacid phenathren-1-ylmethyl ester, 2H-1,4-thiazine, 4H-1,4-thiazine,2H-1,3-thiazine, 1-thia-4-oxacyclohexa-2,5-diene (1,4-oxathiine),1,3,4-oxadiaazole, 4H-1,3,5-oxadiazine, 4H-1,3,4,5-oxatriazine, and1-oxa-4-az-cyclohept-2-ene (4,5,6,7-tetrahydro-1,4-oxazepine) rings.

Any of the monocyclic rings, A, described above, may be fused to 1 to 3additional 4 to 8 member rings, as long as ring A has two adjacentpositions The additional rings are fused either directly to ring A or toeach other.

The additional 4 to 8 member fused rings include all of the ringspreviously described for ring A. However, the additional rings alsoinclude 4 and 8 member rings not included for ring A. For example, somecarbocyclic 4 and 8 member rings include cyclobutane, cyclooctane,cyclobutene, cyclooctene, 1,3-cyclooctadiene, 1,5-cyclooctadiene, and1,3,5,7-cyclooctatetraene rings.

Some examples of additional saturated heterocyclic 4 and 8 member ringsinclude azetidine (1-azacyclobutane or trimethylene imine), oxetane(1-oxacyclobutane or trimethylene oxide), thietane (1-thiacyclobutane ortrimethylene sulfide), azocane (1-azacyclooctane or heptamethyleneimine), oxocane (1 -oxacyclooctane or heptamethylene oxide), andthiocane (1-thiacyclooctane or heptamethylene sulfide) rings. Someexamples of additional unsaturated heterocyclic 4 and 8 member ringsinclude 1-thiacyclobut-2-ene (2H-thiete), 1-oxacyclobut-2-ene(2H-oxete), 1-azacyclobut-2-ene (1,2-dihydroazete),1-thiacycloocta-2,4,6-triene (2H-thiocine), 1-oxacycloocta-2,4,6-triene(2H-oxocine), and 1-azacycloocta-2,4,6-triene (2H-azocine) rings.

The fused rings may be all carbocyclic, and independently, saturated orunsaturated. Some examples of a carbocyclic ring fused to one 4 to 8member carbocyclic ring, wherein all of the carbocyclic rings aresaturated, include bicyclo[3.3.0]octane (octahydropentalene),bicyclo[4.3.0]nonane (octahydroindene), bicyclo[4.4.0]decane(decahydronaphthalene), bicyclo[6.3.0] undecane(decahydrocyclopentacyclooctene or decahydrocyclopenta[8]annulene), andbicyclo[4.2.0]octane rings.

Some examples of carbocyclic rings fused to two or three 4 to 8 membercarbocydclic rings, wherein the carbocyclic rings are all saturated,include tetradecahydroanthracene, tetradecahydrophenanthrene,octadecahydrotriphenylene, dodecahydrobiphenylene,hexadecahydrodibenzo[a,e]cyclooctene, eicosahydrotribenzo[a,c,e]cyclooctene, octadecahydro-2,3-benzanthracene(octadecahydronaphthacene), hexadecahydrocyclopenta[a]phenanthrene,octadecahydro-1,2-benzanthracene (octadecahydro-benzo[a]anthracene),hexadecahydropyrene, and octadecahydrochrysene rings.

Alternatively, the fused carbocyclic rings may be composed of one or tworings that are saturated and one or two rings that are unsaturated. Someexamples of a carbocyclic ring fused to 1 to 3 additional carbocyclic 4to 8 member rings, wherein at least one of the rings is saturated and atleast one of the other rings is unsaturated includebicyclo[4.3.]non-3-ene, bicyclo[4.3.0]non-7-ene,bicyclo[4.4.0]dec-8-ene, and bicyclo[4.4.0]dec-7,9-diene rings.

Alternatively, all of the fused carbocyclic rings mail be unsaturated.Some of the unsaturated carbocyclic rings may also be aromatic. Someexamples of a carbocyclic ring fused to 1 to 3 additional 4 to 8 membercarbocyclic rings, wherein all of the carbocyclic rings are unsaturated,include naphthalene, phenanthrene, anthracene, triphenylene, azulene,chrysene, pyrene, biphenylene, bicyclo[4.3.0]non-3-ene,bicyclo[4.3.0]non-7-ene, bicyclo[4.4.0]dec-8-ene,bicyclo[4.4.0]dec-1-ene, bicyclo[4.4.0]dec-7.9-diene,bicyclo[4.3.0]nona-2,4,7-triene, bicyclo[4.4.0]deca-2,4,7,9-tetraene,bicyclo[4.4.0]deca-1,3,8-triene, 1,4,5,8-tetrahydronaphthalene,cyclopentacyclooctene (cyclopenta[8]annulene), dibenzo[a,e]cyclooctene,tribenzo[a,c,e]cyclooctene, 2,3-benzanthracene, 1,2-benzanthracene, andcyclopenta[a]phenanthrene rings.

In another embodiment, the carbocyclic ring may be fused to 1 to 3additional heterocyclic 4 to 8 member rings. Some examples of suchcompounds include indoline, 1-acetylindoline, 2,3-benzofuran,thianapthene, quinoline, isoquinoline, phthalazine, decahydroquinoline,cyclopentyl[b]pyridine (6,7-dihydro-5H-[1]pyrindine), benzimidazole,benzothiazole, benzisoxazole, benzodioxole, quinoxaline, quinazoline,benzoxazine, cinnoline, 2-cyclopenten-1-one ethylene ketal,1,4-cyclohexanedione bis(ethylene ketal), benzofurazan,1,10-phenanthroline, 4,7-phenanthroline, 1,7-phenanthroline,2,1,3-benzothiadiazole, benzofuroxan, benzotriazole,1-acetylbenzotriazole,6,7-dihydro-5H-cyclohepta[2,1-b;3,4-b′]dipyridine, benzo[b]oxepine,benzo[b]azepine, benzo[b]oxocine, benzo[b]azocine, and7-oxa-bicyclo[4.2.0]octa-1,3,5-triene rings.

In a further embodiment, the carbocyclic ring may be fused to 2 or 3additional 4 to 8 member rings, which are a mixture of carbocyclic andheterocyclic rings. Some examples of such compounds include acridine,phenazine, 5,1 0-dihydro-5,10-dimethylphenazine, phenanthridine,9H-carbazole, dibenzofuran, xanthene, dibenzothiophene, phenoxazine,phenothiazine, phenoxathiin, dibenzo[b]azepine,5H-dibenzo[b]azepine-5-carboxamide, 6,11-dihydrodibenzo[b,e]oxepine, andbenz[a]phenoxazine, and 6,11-dihydrodibenzo[b,e]thiepine rings.

In a different embodiment, ring A is a heterocyclic ring fused to 1 to 3additional 4 to 8 member rings that are also heterocyclic. Some examplesof such compounds include 1,3,5,8-tetraazanaphthalene (pteridine),1,2,4-triazolo[1,5-a]pyrimidine, 1H-1,2,3-triazolo[4,5]pyridine,1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, 7H-purine,1,2,4-triazolo[4,3-a]-1,3,5-triazine (s-triazolo[4,3-a]-s-triazine),1,3,8-triazanaphthalene (pyrido[2,3-d]pyrimidine, 1,8-naphthyridine,1,8,9-triazaanthracene, 1,5-diazabicyclo[4.3.0]non-5-ene(2,3,4,6,7,8-hexahydro-pyrrolo[1,2-a]pyrimidine),1,8-diazabicyclo[5.4.0]undec-7-ene(2,3,4,6,7,8,9,10-octahydro-pyrimido[1,2-a]azepine),2,3-dihydrothieno[3,4-b]-1,4-dioxin, 4H-1,3-oxathiolo[5,4-b]pyrrole, andthieno[3,2-b]furan rings.

Ring A and the additional rings are unsubstituted or substituted with 1to 4 substituents. Some suitable substituents include lower alkyl, aryl,heteroaryl, hydroxy-(lower alkyl), amino-(lower alkyl), N-(loweralkyl)amino-(lower alkyl), N,N-di(lower alkyl)amino-(lower alkyl),N-arylamino-(lower alkyl), N,N-diarylamino-(lower alkyl),N-(heteroaryl)amino-(lower alkyl), N,N-di(heteroaryl)amino-(loweralkyl), hydroxylamino, O-(lower alkoxy)amino, O-aryloxyamino,O-heteroaryloxyamino, fluoro, chloro, bromo, nitro, hydroxyl, loweralkoxy, aryloxy, carboxyl (hydroxycarbonyl), lower alkanoyl, loweralkanoyloxy, amino, N-(lower alkyl)amino, N,N-di(lower alklyl)amino,formamido (formylarnino), N-acylamino, N,N-diacylamino (imido),hydrazido, N-(lower alkyl)hydrazido, N,N-di(lower alkyl)hydrazido,N-arylhydrazido, N,N-diarylhydrazido, N-(heteroaryl)hydrozido,N,N-di(heteroaryl)hydrazido, carboxamido (carbamoyl), N-(loweralkyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl,N,N-diarylcarbamoyl, N-heteroarylcarbamoyl, N,N-di(heteroaryl)carbamoyl,hydroxysulfonyl (sulfonic acid), (lower alkoxy)sulfonyl, aryoxysulfonyl,heroaryloxysulfonyl, hydroxysulfonyl-(lower alkyl), (loweralkoxy)sulfonyl-(lower alkyl), aryoxysulfonyl-(lower alkyl),heroarN,loxysulfonyl-(lower alkyl), (lower alkane)sulfonyl,arenesulfonyl, or heteroarenesulfonyl.

In this specification, various substituents are defined as being“lower,” which means having 1 to 6 carbon atoms. Lower alkyl groups maybe branched or unbranched. Some examples of lower alkyl groups includemethyl, ethyl, n-propyl, isopropyl n-butyl, s-butyl, i-butyl, t-butyl,n-pentyl, n-hexyl, 4-methyl-2-pentyl, etc. The lower alkyl group may beunsubstituted or substituted at any position with an aryl, heteroaryl,fluoro, chloro, or bromo group. Lower alkanoyl and lower alkoxy groupscontain a lower alkyl portion as defined above, and are unsubstituted orsubstituted with 1 to 4 groups independently selected from aryl,heteroaryl or fluoro.

Y and Z are substituents at adjacent positions (e.g., 1, 2; ortho) onring A. Y represents:

Each of R¹ and R² independently represents H, lower alkyl, aryl,heteroaryl, fluoro, chloro, bromo, lower alkoxy (i.e., alkyloxy),aryloxy, heteroaryloxy, N,N-di(lower alkyl)amino, N,N-diarylamino,N,N-di(heteroaryl)amino, (lower alkyl)thio, arylthio, heteroarylthio,(lower alkane)sulfinyl, arenesulfinyl, heteroarenesulfinyl, (loweralkyl)sulfonyl, arenesulfonyl, heteroarenesulfonyl, (loweralkoxy)-(lower alkyl), aryloxy-(lower alkyl), heteroaryloxy-(loweralkyl), N,N-di(lower alkyl)amino-(lower alkyl), N,N-diarylamino-(loweralkyl), N,N-di(heteroaryl)amino-(lower alkyl), (lower alkyl)thio-(loweralkyl), arylthio-(lower alkyl), heteroarylthio-(lower alkyl), (loweralkane)sulfinyl-(lower alkyl), arenesulfinyl-(lower alkyl),heteroarenesulfinyl-(lower alkyl), (lower alkyl )sulfonyl-(lower alkyl),arenesulfonyl-(lower alkyl), aromatic acyl is benzoyl; (loweralkane)sulfonyl is methanesulfonyl or trifluoromethanesulfonvl;arenesulfonyl is benzenesulfonyl or p-toluenesulfonyl.

In one embodiment, G is hydrogen, a metal ion, a quaternary ammoniumion, or lower alkyl. The metal ion may be any positively charged metalion. Some examples of suitable metal ions include Li⁺, Na⁺, and K⁺. Someexamples of suitable quaternary ammonium ions include NH₄ ⁺, HNEt₃ ⁺,hydrogen-pyridinium ion, N-methylpyridiniuim ion, andN-methylmorpholinium ion.

In another embodiment, X-G represents a carbonyl-activating group forthe formation of conjugates between the compounds of the invention and adrug. The activating group is any group that makes the carbonyl groupsandwiched by C_(b) and X of the Y component of the invention more proneto react with a nucleophile. Some examples of activating groups X-Ginclude hydroxybenzotriazole-1-oxy, succinimide-N-oxy, p-nitrophenyloxy,and pentafluorophenyloxy radical. Some examples of nucleophiles includealcohols, thiols, and amines.

In the reaction, the nucleophiles displace the carbonyl-activating groupX-G. For example, the reaction of a drug or other chemical compoundcontaining an alcohol group with the activated ester results in a newester bond with said drug or other chemical compound, the reaction of adrug or other chemical compound containing a thiol group with theactivated ester results in a thioester bond With said drug or otherchemical compound. and the reaction of a drug or other chemical compoundcontaining an amine group with the activated ester results in an amidebond with said drug or other chemical compound.

In another embodiment, G comprises a pharmaceutically active chemicalcompound (e.g. a drug), or the precursor thereof Some examples ofpharmaceutically active chemical compounds or the precursors thereof,include antitumor drugs, antiangiogenic drugs, multi-drug reversalagents, anti-inflammatory drugs, antibiotics, antibacterial agents,antiparasitic drugs, and analgesics. For the purposes of the invention,the parent pharmaceutically active chemical compound or precursorpossesses at least one nucleophilic group capable of reacting with thecarboxyl or heteroarenesulfonyl-(lower alkyl), (lower alkoxy)carbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, carboxamido (carbamoyl),N-(lower alkyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl,N,N-diarylcarbamoyl, N-heteroarylcarbamoyl orN,N-di(heteroaryl)carbamoyl.

Each of R³ and R⁴ independently represents H, lower alkyl, aryl,heteroaryl, fluoro, lower alkoxy, aryloxy, heteroaryloxy, N,N-di(loweralkyl)amino, N,N-diarylamino, N,N-di(heteroaryl)amino, (loweralkyl)thio, arylthio, heteroarylthio, (lower alkane)sulfinyl,arenesulfinyl, heteroarenesulfinyl, (lower alkyl)sulfonyl,arenesulfonyl, heteroarenesulfonyl, (lower alkoxy(-(lower alkyl),aryloxy-(lower alkyl), heteroaryloxy-(lower alkyl), N,N-di(loweralkyl)amino-(lower alkyl), N,N-diarylamino-(lower alkyl),N,N-di(heteroaryl)amino-(lower alkyl), (lower alkyl)thio-(lower alkyl),arylthio-(lower alkyl), heteroarylthio-(lower alkyl), (loweralkane)sulfinyl-(lower alkyl), arenesulfinyl-(lower alkyl),heteroarenesulfinyl-(lower alkyl), (lower alkyl)sulfonyl-(lower alkyl),arenesulfonyl-(lower alkyl), heteroarenesulfonyl-(lower alkyl), (loweralkoxy)carbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, carboxamido(carbamoyl), N-(lower alkyl)carbamoyl or N,N-(lower alkyl)carbamoyl,N-arylcarbamoyl, N,N-diarylcarbamoyl, N-heteroarylcarbamoyl orN,N-di(heteroaryl)carbamoyl.

R¹ and R² or R³ and R⁴ are optionally connected to form a 3 to 8 memberring. The ring may be cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, aryl, or heteroaryl. Examples of such rings havebeen previously given.

X and E independently represent O, S, NR^(a) or NR^(b). R^(a) and R^(b)independently represent H, lower alkyl, cycloalkyl, heterocycloalkyl,cycloalkenyl, heterocycloalkenyl, aryl, aryl lower alky)l, heteroaryl,lower alkanoyl, aromatic acyl (arenecarbonyl), heteroaromatic acyl(heteroarenecarbonyl), (lower alkoxy)carbonyl, aryloxycarbonyl,heteroaryloxycarbonyl, N-arylcarbamoyl, N,N-diarylcarbamoyl,N-heteroarylcarbamoyl, N,N-di(heteroaryl)carbamoyl, (loweralkane)sulfonyl, arenesulfonyl, or heteroarenesulfonyl.

In preferred embodiments, (lower alkoxy)carbonyl is tert-butoxycarbonyl,benzyloxycarbonyl or phenanthrylmethoxycarbonyl; lower alkanoyl isacetyl; thiocarboxyl group or activated ester thereof, of the compoundsof the invention, prior to becoming G.

Z represents:

R¹, R², R³, R⁴, and E in Z are defined as given previously for Y. Thesubscript v represents 0 or 1, provided that when v is 0, then J ishydrogen, a metal ion, or a quaternary ammonium ion; and X is O and G isH.

In one embodiment, J is a lower alkayl, aryl or heteroaryl group. Someexamples of preferred groups include methyl, phenyl, p-fluorophenyl,p-nitrophenyl, o-nitrophenyl, and pyridine-2-yl groups. J groups such asp-nitrophenyl, o-nitrophenyl, or pyridine-2-yl are known to hasten thethiol exchange reaction, and may be regarded as disulfide-activatinggroups.

In another embodiment, J is a substituent that contains an activatedester, or a precursor thereof, and thus, permits a specific bindingagent, such as an antibody, to bind to the disulfide end of the Zcomponent without undergoing a thiol exchange process. Some classes ofsuch J substituents include omega-hydroxycarbonyl-(lower alkyl),omega-(lower alkoxy)carbonyl-(lower alkyl), oromega-(X-G)-carbonyl-(lower alkyl) group, wherein the X-G activatinggroup is defined above. Some suitable examples of such groups include2-hydroxycarbonylethyl, 2-methoxycarbonylethyl,p-nitrophenyloxycarbonylpropyl, pentafluorophenyloxycarbonylbutyl,2-(succiminide-N-oxycarbonyl)-1-methylpropyl. As an example, anactivated ester substituent J reacts with the lysine residues of amonoclonal antibody, another specific binding protein or a specificbinding peptide to form a conjugate.

In another embodiment, J is a specific binding agent. A specific bindingagent is a protein, peptide, lectin, saccharide, or other moiety thatselectively binds to a molecule on the surface of a cell. Some types ofmolecules on the surface of a cell that may be targeted by a specificbinding agent include receptors, oligosaccharides, lectins, adhesionmolecules, proteoglycams, integrins, immunoglobulins, majorhistocompatibility complex, e.g., human leukocyte antigen, andglycoproteins. Some examples of receptors include tyrosine kinasereceptors, such as vascular endothelial growth factor (VEGF) receptor,and epidermal growth factor (EGF) receptors, e.g., HER-1, HER-2, HER-3,and HER-4.

A specific binding agent may, for example, be a receptor-specificligand. A receptor-specific ligand is a natural or synthetic molecule,such as a hormone or neurotransmitter, which specifically binds to areceptor on the surface of a cell. Some examples of receptor-specificligands include bombesin, transferrin, VEGF, and EGF.

In another embodiment, the specific binding agent is a molecule thatcomprises the hypervariable region (CDR) of an antibody and has bindingcharacteristics that are the same as, or comparable to, those of thewhole antibody. Preferably, the specific binding agent is an antibody ora functional equivalent of an antibody, such as a fragment of anantibody. More preferably, the antibody is a monoclonal antibody or afunctional equivalent derived from a monoclonal antibody.

Suitable fragments of antibodies include any fragment that comprises asufficient portion of the hypervariable region to bind specifically, andwith sufficient affinity, to a molecule on the surface of a cell. Suchfragments may, for example, contain one or both Fab fragments, or theF(ab′)₂ fragment. Preferably, the antibody fragments contain all sixcomplementarity determining regions of the whole antibody, althoughfunctional fragments containing fewer than all of such regions, such asthree, four or five CDRs, may also be suitable.

The preferred fragments are single chain antibodies, or Fv fragments.Single chain antibodies are polypeptides that comprise at least thevariable region of the heavy chain of the antibody linked to thevariable region of the light chain: with or without an interconnectinglinker. These chains may be produced in bacteria or in eucaryotic cells.

The antibodies and functional equivalents may be members of any class ofimmunoglobulins, such as: IgG, IgM, IgA, IgD, or IgE, and the subclassesthereof The preferred antibodies are members of the IgGl subclass. Thefunctional equivalents may also be equivalents of combinations of any ofthe above classes and subclasses.

Suitable variable and hypervariable regions of antibodies may be derivedfrom antibodies produced by any mammal in which monoclonal antibodiesare made. Some examples of suitable mammals include rabbits, rats, mice,horses, goats, and primates. Preferably, the monoclonal antibodies arederived from mice. The monoclonal antibodies thus obtained are humanizedby methods known in the art for the purpose of human clinical use.

In Y and Z, each of a, b, c, d, e and f independently represents 0 or 1,with the provisions that a+c equals 0, 1, or 2; b+d equals 0, 1, or 2;a+b+c+d+e+f equals 1, 2, or 3; additionally provided that when f is 1,then d is 1, and when d is 0, then f is 0; and when both e and b are 0,then neither R¹ nor R² is chloro or bromo.

The compounds of the present invention may be linked to apharmaceutically active chemical component G, or a precursor thereof, bymethods known in the art. For example, the carbonyl group in thecompound may first be reacted with a suitable activating compound toattach an activating group X-G. Some examples of activating compounds,or a combination of activating compounds, capable of attaching anactivating group X-G onto the compounds of the invention, include thecombination of 1,3-diisopropylcarbodiimide (DIC) or1,3-dicyclohexylcarbodiimide (DCC) with 1-hydroxybenzotriazole;N-hydroxysuccinimide; and p-(N,N-dimethylamino)pyridine (DMAP). Theresulting activated ester of the compound is reacted with, for example,a drug or pro-drug bearing a suitable nucleophilic group or groups, asdescribed above. The drug thereby becomes linked to the carboxyl end ofthe Y component of the compound.

Similarly, the compounds of the present invention may be linked to aspecific binding agent by methods known in the art. The disulfide groupin component Z will undergo the well known thiol exchange process in thepresence of a specific binding agent bearing at least one thiol group. Adisulfide-activating group is not required under all conditions, but maybe used to hasten the reaction. The specific binding agent therebybecomes linked to the compound through a disulfide group on the Zcomponent.

Alternatively, a specific binding agent may be modified to containactivated disulfide linkages in order to react Faith the reduced thiolform of the Z component when Y possesses a carboxylic acid terminus,i.e., when v is 0 and J is hydrogen, a metal ion, or a quaternaryammonium ion, and X is O and G is H The disulfide-activated specificbinding agent binds to the reduced thiol form of Z through the disulfideexchange process.

The modification of specific binding agents, such as monoclonalantibodies, with an acyl group bearing an activated disulfide moiety iswell known in the art. [See R. V. J. Chari, Advanced Drug DeliveryReviews, 1998, 31, 89-104; I. Ojima, X. Geng, X. Wu, C. Qu, C. P.Borella, H. Xie, S. D. Wilhelm, B. A. Leece, L. M. Bartle, V. S.Goldmacher, and R. V. J. Chari, J. Med. Chem. 45, 5620-5623 (2002)].Specific binding proteins such as monoclonal antibodies bearing thiolgroups can be readily generated from activated disulfide derivatives byappropriate reducing agents known in the art. [See, for example, Trail,P. A.; Willner, D.; Knipe, J.; Henderson, A. J.: Lasch, S. J.; Zoeckler,M. E.; TrailSmith, M. D.; Doyle, T. W.; King, H. D.; Casazza, A. M.;Braslawsky, G. R.; Brown, J.; Hofstead, S. J.; Greenfield, R. S.;Firestone, R. A.; Mosure, K.; Kadow, K. F.; Yang, M. B.; Hellstroem, K.E.; Hellstroem, I. “Effect of linker variation on the stability,potency, and efficacy of carcinoma-reactive BR64-doxorubicinimmunoconjugates”, Cancer Res. 1997, 57, 100-105, wherein dithiothreitol(DTT) was used as reducing agent.]

The resulting conjugate containing the specific binding agent may befurther coupled to, for example, a drug, bearing a hydroxyl, thiol, oramine moiety through the condensation reaction between the carboxylicacid terminus in the Y component of the conjugate via an activatedester, as described above.

In a preferred embodiment, G is a pharmaceutically active chemicalcompound, preferably a drug, and J is a specific binding agent,preferably a molecule comprising the hypervariable region (CDR) of anantibody. The above combination of G and J forms a linker drugconjugate.

Although not bound by any theory, the invention is believed to work asfollows. First, the linker drug conjugate binds to a cell by theattachment of the specific binding agent. The linker drug conjugate isinternalized into the cell through, for example, endocytosis. Onceinside the cell, the disulfide bond of the linker drug conjugateundergoes cleavage by an endogenous peptide that cleaves disulfidebonds, e.g., glutathione (GT). Glutathione is found in most mammaliancells and its level is considerably elevated in hypoxic cells such astumor cells. The cleavage of the disulfide bond causes the release ofthe specific binding agent. Simultaneously, a thiolactonization processoccurs between the remaining sulfur atom and the ester. thioester oramide linkage of the drug. The end result of the thiolactonizationprocess is the release of the original and the most active form of thedrug, inside of the targeted cell. The reaction scheme below is providedfor further elucidation of the process.

The linker drug conjugate is either uncharged or in the form ofpharmaceutically acceptable salts. The term “pharmaceutically acceptablesalt” refers to a salt prepared from a suitable linker drug conjugateand, for example, an acid or a base. The salt is acceptably non-toxicand has acceptable pharmacokinetics.

Such salts are formed by well known procedures. Suitable acids forproducing salts of the linker drug conjugates include mineral acids andorganic acids. Some examples of mineral acids include hydrochloric,hydriodic, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuricacids. Some examples of organic acids include tartaric, acetic, citric,malic, benzoic, pyridine, gluconic, gulonic, succinic, arenesulfonic,e.g. p-toluenesulfonic acids, and the like.

Suitable bases for producing salts of the linker drug conjugates includeinorganic bases and organic bases. Some examples of inorganic basesinclude hydroxides of lithium, sodium, potassium, magnesium and calcium.Some examples of organic bases include primary, secondary, and tertiary,alkyl amines

For the pharmaceutical purposes described above, the linker drugconjugate of the invention can be formulated in pharmaceuticalpreparations optionally including a suitable pharmaceutical carrier(vehicle) or excipient. In this specification, a pharmaceutical carrieris considered synonymous with a vehicle or an excipient as understood bypractitioners in the art. Examples of carriers include starch, milk,sugar, certain types of clay, gelatin, stearic acid or salts thereof,magnesium or calcium stearate, talc, vegetable fats or oils, gums andglycols.

The linker drug conjugate formulation may also comprise one or more ofthe following: a stabilizer, a surfactant, a salt, a buffering agent, ora combination thereof. The stabilizer may be, for example, an aminoacid, such as glycine; or an oligosaccharide, such as sucrose,tetralose, lactose or a dextran. Alternatively, the stabilizer may be asugar alcohol, such as mannitol; or a combination thereof.

Some examples of suitable surfactants include Tween 20, Tween 80; apolyethylene glycol or a polyoxyethylene polyoxypropylene glycol, suchas Pluronic F-68 at from about 0.001% (w/v) to about 10% (w/v). The saltor buffering agent may be any salt or buffering agent, such as, forexample, sodium chloride, or sodium/potassium phosphate, respectively.

The linker drug conjugate formulation may additionally contain one ormore conventional additives. Some examples of such additives include asolubilizer such as, for example, glycerol; an antioxidant such as, forexample, benzalkonium chloride (a mixture of quaternary ammoniumcompounds, known as “quart”), benzyl alcohol. chloretone orchlorobutanol; an anaesthetic agent such as, for example, a morphinederivative; an isotonic agent, or a combination of these. As aprecaution against oxidation or other spoilage, the linker drugconjugate formulation may be stored under nitrogen gas in vials sealedwith impermeable stoppers.

For aqueous suspensions, emulsifying agents, suspending agents, or acombination thereof, may be added. In addition, coloring, sweetening,and flavoring agents may be added to the formulation. Sterile solutionsof the linker drug conjugates can also be employed. If required, the pHof the solutions can be suitably adjusted and buffered.

The linker drug conjugates ma:, be administered alone or as an adjunctwith other conventional drugs for treating conditions or diseases,including cancer. The linker drug conjugates may be administered by anymethod known in the art. Some examples of suitable modes ofadmininistration include oral, systemic, and topical administration.

Liquid or solid oral formulations are known in the art. Some examples offormulations suitable for oral administration include tablets, capsules,pills, troches, elixirs, suspensions, and syrups.

Systemic administration includes enteral or parenteral modes ofadministration, e.g., intravenous; intramuscular; subcutaneous; orintraperitoneal. For example, the linker drug conjugate formulation maybe administered by injection of a solution or suspension; orintranasally, in the form of, for example, a nebulizer, liquid mist, orintranasal spray; or transdermally, in the form of, for example, apatch; or rectally, in the form of, for example, a suppository; orintrabronchially, in the form of, for example, an inhaler spray.

The timing of the administration of the linker drug conjugateformulation may also be modified. For example, the formulation may beadministered intermittently or by controlled release. Controlled releaseadministration is a method of drug delivery to achieve a certain levelof the drug over a particular period of time.

Examples have been set forth below for the purpose of illustration andto describe the best mode of the invention at the present time. However,the scope of this invention is not to be in any wav limited by theexamples set forth herein.

EXAMPLE 1 Preparation of (2-mercaptophenyl)Acetic Acid

2-Oxo-2,3-dihydrobenzo[b]thiophene was prepared by the literature method(Lumma, W. C., Jr.; Dutra, G. A.; Voeker, C. A. J. Org. Chem. 1970, 35,3442-3444; Bordwell, F. G.; Fried, H. E. J. Org. Chem. 1991, 56,4218-4223) from commercially available benzo[b]thiophene (AldrichChemicals Co.) in high yield. 2-oxo-2,3-dihydrobenzo[b]thiophene (457mg, 3.04 mmol), thus obtained, was added to a solution of KOH (1 N, 15mL) and THF (5 mL). The solution was stirred at 60° C. for 14 h, cooledto ambient temperature, and acidified to pH 2 with 5 N hydrochloricacid. The reaction mixture was extracted with CH₂Cl₂ three times. Theorganic layers were combined, dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo, affording 449 mg (88% yield) of the(2-mercaptophenyl)acetic acid product in the form of a pale yellow waxysolid: ¹H NMR (400 MHz, CDCl₃) δ (ppm): 3.53 (s, 1 H), 3.85 (s, 2 H),7.22 (m, 3 H), 7.45 (m, 1 H), 11.82 (broad s, 1 H).

EXAMPLE 2 Preparation of (5-fluoro-2-mercaptophenyl)Acetic Acid

By the same method as described in Example 1,(5-fluoro-2-mercaptophenyl)acetic acid was prepared from5-fluoro-3H-benzo[b]thiophen-2-one in 95% yield in the form of a paleyellow solid: ¹H NMR (400 MHz, CDCl₃) δ (ppm): 3.40 (s, 1 H), 3.84 (s, 2H), 6.92 (ddd, J=8.4 Hz, 8.4 Hz, 2.8 Hz, 1 H), 7.00 (dd,J=9.2 Hz, 2.8 Hz1 H), 7.42 (dd,J=8.4 Hz, 5.6 Hz, 1 H). 11.41 (s, broad, 1 H); ¹⁹F NMR(282 MHz, CDCl₃) δ (ppm): −113.05

EXAMPLE 3 3-(2-Mercaptophenyl)-3-Methylbutanoic Acid

By the same method as described in Example1,3-(2-mercaptophenyl)-3-methylbutanoic acid was prepared from4,4-dimethyl-2-oxo-3,4-dihydro-2H-1-benzothiopyran in 85% yield in theform of a pale yellow solid: ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 1.58 (s,6H), 3.12 (s, 2H), 3.66 (s, 1H), 7.2-7.4 (m 4H), 10.2 (broad, 1H).

EXAMPLE 4 Preparation of (2-Methyldisulfanylphenyl)Acetic Acid

To a solution of (2-mercaptophenyl)acetic acid (449 mg) in H₂O (15 mL)and ethanol (7 mL) was added methyl methanethiosulfonate (372 mg, 2.93mmol). After stirring for 20 hours, the reaction mixture was dilutedwith ether and washed with 1 N KOH twice. The water layers were combinedand acidified to pH 2 with 5 N hydrochloric acid. This solution wasextracted with CH₂Cl₂ three times. The organic lavers were combined,dried over anhydrous MgSO₄, filtered, and concentrated in vacuo toafford 576 mg (100% yield) of the (2-methyl disulfanylphenyl)acetic acidproduct in the form of a yellow oil: ¹H NMR (400 MHz, CDCl₃) δ (ppm):2.42 (s, 3 H), 3.92 (s, 2 H), 7.10-7.40 (m, 3 H), 7.80 (d, J=7.6 Hz, 1H), 10.34 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 22.7, 38.9, 127.8,128.1, 128.3, 128.8, 130.4, 130.8, 130.9, 177.1. HRMS (EI): m/e calcd.for C₉H₁₀O₂S₂: 214.012223, Found: 214.012637 (Δ=−1.9 ppm).

EXAMPLE 5 (5-Fluoro-2-Methyldisulfanylphenyl)Acetic Acid

By the same method as described in Example 4,(5-fluoro-2-methyldisulfanylphenyl) acetic acid was prepared from(5-fluoro-2-mercaptophenyl)acetic acid in 74% yield in the form of alight yellow solid: ¹H NMR (400 MHz, CDCl₃) δ (ppm): 2.43(s, 3 H), 3.94(s, 2 H), 7.05 (m, 2 H), 7.74 (m, 1 H), 10.93 (s, broad, 1 H); ¹⁹F NMR(282 MHz, CDCl₃) δ (ppm): −113.06

EXAMPLE 6 3-Methyl-3-(2-Methyldisulfanylphenyl)Butanoic Acid

By the same method as described in Example 4,3-methyl-3-(2-methyldisulfanylphenyl) butanoic acid was prepared from3-(2-mercaptophenyl)-3-methylbutanoic acid in 75% yield in the form ofan oil: ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 1.58 (s, 6H), 2.43 (s, 3H),3.14 (s, 2H), 7.1-7.4 (m, 3H), 7.94 (d, J=7.8 Hz, 1 H), 10.4 (broad,1H),¹³C-NMR (CDCl₃, 75 MHz) 5 (ppm): 22.6, 29.4, 38.4, 44.5, 126.9,127.2, 127.4, 130.5, 135.4, 145.7, 176.6.

EXAMPLE 7 3-Methyl-3-[2-(Pyridine-2-yldisulfanyl)phenyl]Butanoic Acid

To a solution of 3-(2-mercaptophenyl)-3-methylbutanoic acid (227 mg) inaqueous THF was added 2.2′-dipyridyl disulfide (363 mg), and the mixturewas stirred for 15 hours at 40° C. The reaction was quenched by 1 Nhydrochloric acid. The reaction mixture was extracted by ethyl acetateat pH 1, and the extract was dried over anhydrous MgSO₄ and concentratedin vacuo. The crude product was purified on a silica gel column toafford 252 mg (75% yield) of the3-methyl-3-[2-(pyridine-2-yldisulfanyl)phenyl]butanoic acid product inthe form of a light yellow solid: mp 122-125° C., ¹H NMR (CDCl₃, 300MHz) δ (ppm): 1.67 (s, 6H), 3.32 (s, 2H), 7.11 (m, 1H), 7.17 (m, 2H),7.38 (m, 1H), 7.5-7.6 (m, 2H), 7.80 (m, 1H), 8.44 (d, J=4.5 Hz, 1 H),10.52 (broad s, 1 H).

EXAMPLE 8 4-Fluorophenyl 3-Methyl-3-(2-Methyldisulfanylphenyl)Butanoate

To a mixture of 3-methyl-3-(2-methyldisulfanylphenyl)butanoic acid,4-fluorophenol (1.1 eq) and 4-(N,N-dimethylamino)pyridine (0.5 eq) inCH₂Cl₂ was added diisopropylcarbodiimide (1.5-2.0 eq) at 0° C. Themixture was stirred overnight at ambient temperature. The precipitatewas filtered off and the filtrate concentrated by rotary evaporation.The crude product was purified on a silica gel column to afford4-fluorophenyl 3-methyl-3-(2-methyldisulfanylphenyl) butanoate as acolorless oil in 62% yield: ¹H NMR (CDCl₃, 300 MHz) δ (ppm): 1.76 (s,6H), 2.52 (s, 3H), 3.44 (s, 2H), 6.70 (dd, J=9.0, 4.2 Hz, 2H), 7.00 (t,J=9.0 Hz, 2 H), 7.2-7.3 (m, 2H), 7.36 (dd, J=7.8, 1.5 Hz, 1H), 7.96 (dd,J=7.8, 1.2 Hz, 1 H). ¹⁹F NMR (CD₃CN, 282 MHz) δ (ppm): −119.2.

EXAMPLE 9 4-Fluorophenyl3-Methyl-3-[2-(Pyridine-2-yldisulfanyl)Phenyl]butanoate

By the same method as described in Example 8, 4-fluorophenyl3-methyl-3-[2-(pyridine2-yldisulfanyl)phenyl]butanoate was prepared inthe form of a colorless viscous oil in 66% yield: ¹H NMR (CDCl₃, 300MHz) δ (ppm): 1.74 (s, 6H), 3.43 (s, 2H), 6.60 (m, 2H), 6.93 (m, 2H),7.07 (m, 1H), 7.20 (m, 2H), 7.42 (m, 2H), 7.59 (d, J=7.8 Hz, 1 H),7.77(m, 1H), 8.46 (d, J=3.6 Hz, 1 H). ¹⁹F NMR (CD₃CN, 282 MHz) δ (ppm):−119. 1.

EXAMPLE 10 Preparation of2′-(2-Methyldisulfanylphenyl)acetyl-3′-Dephenyl-3′-(2-Methylpropyl)-10-Propanoyldocetaxel

To a solution of 3′-dephenyl-3′-(2-methylpropyl)-10-propanoyldocetaxel(12.6 mg, 0.015 mmol) and (2-methyldisulfanylphenyl)acetic acid (6.7 mg,0.031 mmol) in CH₂Cl₂ (2 mL) was added diisopropylcarbodiimide (3.8 mg,0.03 mmol) and 4-(N,N-dimethylamino)pyridine (1.8 mg, 0.015 mmol). Thereaction mixture was stirred for 2.5 hours, and then concentrated invacuo. Purification of the crude product by column chromatography onsilica gel using hexans/EtOAc (3/1 to 2/1) as eluent afforded 13.2 mg(85% yield) of the2′-(2-methyldisulfanylphenyl)acetyl-3′-dephenyl-3′-(2-methylpropyl)-10-propanoyldocetaxel product in the form of a white solid; ¹H NMR (400MHz, CDCl₃)δ (ppm): 0.92 (m, 6 H), 1.13 (s, 3 H), 1.22-1.27 (m, 6 H),1.30 (s, 9 H), 1.67 (s, 3 H), 1.87 (m, 3 H), 1.88 (s, 3 H), 2.36 (s, 3H), 2.40 (s, 2 H), 2.44 (s, 3 H), 2.46 (m, 1 H), 2.54 (m, 2 H), 3.80 (d,J=7.2 Hz, 1 H), 3.92 (d, J=16.4 Hz, 1 H), 4.06 (d, J=16.4 Hz, 1 H), 4.18(d, J=8.4 Hz, 1 H), 4.29 (m, 2 H), 4.42 (dd, J=10.2, 6.7 Hz, 1 H), 4.58(d, J=10.4 Hz, 1 H), 4.90 (d, J=2.8 Hz, 1 H) (H₂), 4.97 (d, J=8.0 Hz, 1H), 5.65 (d, J=7.0 Hz, 1 H), 6.19 (m, 1 H), 6.29 (s, 1 H), 7.30 (m, 3H), 7.47 (t, J=7.6 Hz, 2 H), 7.59 (t, J=7.6 Hz, 1 H), 7.77 (d, J=7.6 Hz,1 H), 8.11 (d, J=7.2 Hz, 2 H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 9.0,9.5, 14.8, 21.9, 22.5, 22.7, 23.0, 24.6, 26.5, 27.5, 28.1, 29.7, 35.4,38.8, 41.1, 43.1, 45.6, 48.9, 58.4.,71.7, 72.1, 74.6, 75.1, 75.4, 76.4,79.3, 79.8, 80.9, 84.4, 128.0, 128.4, 128.6, 129.2, 130.2, 130.3, 131.0,132.3, 133.5, 143.4, 155.3, 163.4, 167.0, 168.2, 169.6, 170.1, 174.6,204.0. HRMS (FAB): m/e calcd. for C₅₃H₆₉NO₁₆S₂H⁺:1040.413605. Found:1040.413600 (Δ=0.0 ppm).

EXAMPLE 11 Preparation of2′-(5-Fluoro-2-Methyldisulfanylphenyl)Acetyl-3′-Dephenyl-3′-(2-Methylpropyl)-10-propanoyldocetaxel

By the same method as described in Example 10,2′-(5-fluoro-2-methyldisulfanylphenyl)acetyl-3′-dephenyl-3′-(2-methylpropyl)-10-propanoyldocetaxel wasprepared from 3′-dephenyl-3′-(2-methylpropyl)- 10-propanoyldocetaxel and(5-fluoro-2-methyldisulfanyl phenyl)acetic acid in 75% yield in the formof a white solid: ¹H NMR (400 MHz, CDCl₃) δ (ppm): 0.94 (m, 6 H), 1.13(s, 3 H), 1.20-1.25 (m, 6 H), 1.30 (s, 9 H), 1.60 (s, 3 H), 1.65 (s, 2H), 1.66 (s, 3 H), 1.90 (s, 3 H), 2.36(s, 3 H), 2.43 (s, 3 H), 2.48 (m,1 H), 2.54 (m, 2 H), 3.80 (d, J=7.2 Hz, 1 H), 3.95 (d, J=16.4 Hz, 1 H),4.08 (d, J=16.4 Hz, 1 H), 4.19 (d, J=8.4 Hz, 1 H), 4.29 (d, J=8.4 Hz, 1H), 4.33 (m, 1 H), 4.44 (dd, J=10.2, 6.7 Hz, 1 H), 4.57 (d, J=10.4 Hz, 1H), 4.91 (d, J=2.0 Hz, 1 H), 4.97 (d, J=8.0 Hz, 1 H), 5.66 (d, J=7.2 Hz,1 H), 6.19 (t, J=8.0 Hz, 1 H), 6.29 (s, 1 H), 7.04 (m, 1 H), 7.10 (dd,J=9.2, 2.8 Hz, 1 H), 7.47 (t, J=7.2 Hz, 2 H), 7.59 (t, J=7.2 Hz, 1 H),7.71 (dd, J=8.4, 5.6 Hz, 1 H), 8.11 (d, J=7.2 Hz, 2 H); ¹⁹F NMR (282MHz, CDCl₃) δ (ppm): −1 12.84.

EXAMPLE 12 Cleavage of the Disulfide Bond in the Drug-Linker Conjugate

In order to confirm that the designed drug-release mechanism works underphysiological conditions, a control experiment was performed using ananticancer linker drug conjugate and cysteine as a model of glutathione.The results of the experiment below provide clear evidence that the drugis released according to the desired mechanism of the invention.

To a solution of the drug-linker conjugate,2′-(5-fluoro-2-methyldisulfanylpbenyl)acetyl-3′-dephenyl-3′-(2-methylpropyl)-10-propanoyldocetaxel, (4.8 mg,0.0045 mmol) in CH3CN (1.1 mL) was added cysteine (2.20 mg, 0.018 mmol)in buffer (pH 7.0, 0.56 mL) solution. The mixture was stirred at roomtemperature. The progress of the disulfide cleavage and drug releaseprocess was monitored by HPLC [Conditions: C-18 column. Flow rate: H₂O,0.6 mL/min; CH₃CN, 0.4 mL/min., ambient temperature] periodically. Theformations of thiolactone (5-fluoro-3H-benzo[b]thiophen-2-one)(retention time=9.05 min) and taxoid(3′-dephenyl-3′-(2-methylpropyl)-10-propanoyldocetaxel) (retentiontime=14.01 min) as well as the disappearance of the drug-linkerconjugate (retention time=18.20 min) was clearly observed. After 2hours, the drug-linker conjugate completely disappeared and the cleanrelease of the taxoid together with the formation of the thiolactone wasconfirmed.

Thus, while there have been described what are presently believed to bethe preferred embodiments of the present invention, those skilled in theart will realize that other and further embodiments can be made withoutdeparting from the spirit of the invention, and it is intended toinclude all such further modifications and changes as come within thetrue scope of the claims set forth herein.

1. A compound having the formula Y-A-Z, wherein: A is a 5, 6, or 7member ring that is monocyclic or is fused to 1 to 3 additional 4 to 8member rings; wherein ring A and, independently, the fused additionalrings are carbocyclic or heterocyclic, and saturated or unsaturated,wherein unsaturated rings are aromatic or non-aromatic; ring A and theadditional rings are unsubstituted or substituted with 1 to 4substituents selected from lower alkyl, aryl, heteroaryl, hydroxy-(loweralkyl), amino-(lower alkyl), N-(lower alkyl)amino-(lower alkyl),N,N-di(lower alkyl)amino-(lower alkyl), N-arylamino-(lower alkyl),N,N-diarylamino-(lower alkyl), N-(heteroaryl)amino-(lower alkyl),N,N-di(heteroaryl)amino-(lower alkyl), hydroxylamino, O-(loweralkoxy)amino, O-aryloxyamino, O-heteroaryloxyamino, fluoro, chloro,bromo, nitro, hydroxyl, lower alkoxy, aryloxy, carboxyl(hydroxycarbonyl), lower alkanoyl, lower alkanoyloxy, amino, N-loweralkyl)amino, N,N-di(lower alkyl)amino, formamido (formylamino),N-acylamino, N,N-diacylamino (imido), hydrazido, N-(loweralkyl)hydrazido, N,N-di(lower alkyl)hydrazido, N-arylhydrazido,N,N-diarylhydrazido, N-(heteroaryl)hydrozido,N,N-di(heteroaryl)hydrazido, carboxamido (carbamoyl), N-(loweralkyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl,N,N-diarylcarbamoyl, N-heteroarylcarbamoyl, N,N-di(heteroaryl)carbamoyl,hydroxysulfonyl (sulfonic acid), (lower alkoxy)sulfonyl, aryoxysulfonyl,heroaryloxysulfonyl, hydroxysulfonyl-(lower alkyl), (loweralkoxy)sulfonyl-(lower alkyl), aryoxysulfonyl-(lower alkyl),heroaryloxysulfonyl-(lower alkyl), (lower alkane)sulfonyl,arenesulfonyl, or heteroarenesulfonyl; Y and Z are substituents atadjacent positions on ring A; Y represents:

Z represents:

X represents O, S, or NR^(a); E represents O, S, or NR^(b); R^(a) andR^(b) independently represent H, lower alkyl, cycloalkyl,heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl, aryl loweralkyl, heteroaryl, lower alkanoyl, aromatic acyl (arenecarbonyl),heteroaromatic acyl (heteroarenecarbonyl), (lower alkoxy)carbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, N-arylcarbamoyl,N,N-diarylcarbamoyl, N-heteroarylcarbamoyl, N,N-di(heteroaryl)carbamoyl,(lower alkane)sulfonyl, arenesulfonyl, or heteroarenesulfonyl; whereinthe lower alkyl, lower alkanoyl and lower alkoxy groups areunsubstituted or substituted with 1 to 4 groups independently selectedfrom aryl, heteroaryl or fluoro; each of a, b, c, d, e and findependently represents 0 or 1; a+c equals 0, 1, or 2; b+d equals 0, 1,or 2; a+b+c+d+e+f equals 1, 2, or 3; provided that when f is 1, then dis 1, and when d is 0, then f is 0; and when both e and b are 0, thenneither R¹ nor R² is chloro or bromo. v represents 0 or 1, provided thatwhen v is 0, then J is hydrogen, a metal ion, or a quaternary ammoniumion; and X is O and G is H; each of R¹ and R² independently representsH, lower alkyl, aryl, heteroaryl, fluoro, chloro, bromo, lower alkoxy(i.e., alkyloxy), aryloxy, heteroaryloxy, N,N-di(lower alkyl)amino,N,N-diarylamino, N,N-di(heteroaryl)amino, (lower alkyl)thio, arylthio,heteroarylthio, (lower alkane)sulfinyl, arenesulfinyl,heteroarenesulfinyl, (lower alkyl)sulfonyl, arenesulfonyl,heteroarenesulfonyl, (lower alkoxy)-(lower alkyl), aryloxy-(loweralkyl), heteroaryloxy-(lower alkyl), N,N-di(lower alkyl)amino-(loweralkyl), N,N-diarylamino-(lower alkyl), N,N-di(heteroaryl)amino-(loweralkyl), (lower alklyl)thio-(lower alkyl), arylthio-(lower alkyl),heteroarylthio-(lower alkyl), (lower alkane)sulfinyl-(lower alkyl),arenesulfinyl-(lower alkyl), heteroarenesulfinyl-(lower alkyl), (loweralkyl)sulfonyl-(lower alkyl), arenesulfonyl-(lower alkyl),heteroarenesulfonyl-(lower alkyl), (lower alkoxy)carbonyl,aryloxycarbonyl, heteroaryloxycarbonyl, carboxamido (carbamoyl),N-(lower alklyl)carbamoyl, N,N-(lower alkyl)carbamoyl, N-arylcarbamoyl,N,N-diarylcarbamoyl, N-heteroarylcarbamoyl orN,N-di(heteroaryl)carbamoyl; each of R³ and R⁴ independently representsH, lower alkyl, aryl, heteroaryl, fluoro, lower alkoxy, aryloxy,heteroaryloxy, N,N-di(lower alkyl)amino, N,N-diarylamino,N,N-di(heteroaryl)amino, (lower alkyl)thio, arylthio, heteroarylthio,(lower alkane)sulfinyl, arenesulfinyl, heteroarenesulfinyl, (loweralkyl)sulfonyl, arenesulfonyl, heteroarenesulfonyl, (lower alkoxy)(loweralkyl), aryloxy-(lower alkyl), heteroaryloxy -(lower alkyl),N,N-di(lower alkyl)amino-(lower alkyl), N,N-diarylamino-(lower alkyl),N,N-di(heteroaryl)amino-(lower alkyl), (lower alkyl)thio-(lower alkyl),arylthio-(lower alkyl), heteroarylthio-(lower alkyl), (loweralkane)sulfinyl-(lower alkyl), arenesulfinyl-(lower alkyl),heteroarenesulfinyl-(lower alkyl), (lower alkyl)sulfonyl-(lower alkyl),arenesulfonyl-(lower alkyl), heteroarenesulfonyl-(lower alkyl), (loweralkoxy)carbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, carboxamido(carbamoyl), N-(lower alkyl)carbamoyl or N,N-(lower alkyl)carbamoyl,N-arylcarbamoyl, N,N-diarylcarbamoyl, N-heteroarylcarbamoyl orN,N-di(heteroaryl)carbamoyl; R¹ and R² or R³ and R⁴ are optionallyconnected to form a 3 to 8 member ring, wherein the 3 to 8 member ringis cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, aryl,or heteroaryl; lower means having 1-6 carbon atoms; either G ishydrogen, a metal ion, a quaternary ammonium ion, lower alkyl, orcomprised of a pharmaceutically active chemical compound or theprecursor thereof; or X-G represents a carbonyl-activating group; J islower alklyl, aryl, heteroaryl, omega-hydroxycarbonyl-(lower alkyl),omega-(lower alkoxy)carbonyl-(lower alkyl), omega-(X-G)-carbonyl-(loweralkyl) group, or comprised of a specific binding agent.